摘要
【目的】明确桂牧一号杂交象草中的主要危害病毒种类及其病毒的发生和群体遗传结构,了解桂牧一号杂交象草该病毒的遗传变异机制。【方法】首先利用Illumina Hiseq 2500平台对桂牧一号杂交象草进行小RNA深度测序,然后利用大肠杆菌重组蛋白表达和RT-PCR技术对其病毒进行检测和鉴定,最后利用生物信息学软件对其病毒进行密码子偏好性的分析和群体遗传结构分析。【结果】桂牧一号杂交象草经小RNA测序检测出烟草花叶病毒;病毒蛋白的大肠杆菌重组表达和病毒基因的RT-PCR结果验证小RNA测序结果的可靠性;桂牧一号杂交象草烟草花叶病毒具有外壳蛋白、运动蛋白、带电蛋白等6个基因,基因总长度为11037 bp,基因平均长度为1839.5 bp,基因间区GC含量为43.35%;桂牧一号杂交象草烟草花叶病毒CP基因的密码子偏好性均较弱,偏好使用以A/T结尾;桂牧一号杂交象草烟草花叶病毒CP基因密码子偏好性的主要影响因素为自然选择,次要影响因素为内部压力;桂牧一号杂交象草烟草花叶病毒CP基因的最优密码子为6个(GAA、GCA、GCC、ACU、UUA和AGA),多以A、U结尾;桂牧一号杂交象草烟草花叶病毒CP基因与重庆烟草分离物Fengjie-1(HE818423.1和HE818424.1)CP基因的核苷酸和氨基酸序列一致性较高。重庆、广西、贵州、湖北、辽宁、四川、云南、韩国、德国、美国10个烟草花叶病毒群体分离物CP基因具有较高的遗传多样性水平,其中美国、韩国和湖北烟草花叶病毒的遗传多样性最高;10个烟草花叶病毒群体分离物CP基因错配曲线图的实际观测值均背离期望值,均出现锯齿状的峰形分布,均处于动态平衡状态,近期均无群体扩张事件;10个烟草花叶病毒群体分离物CP基因的遗传分化明显(F_(ST)>0.25),基因交流频繁(Nm>1);10个烟草花叶病毒群体分离物CP基因的dN/dS均远小于1。【结论】桂牧一号杂交象草烟草花叶病毒与重庆烟草分离物Fengjie-1(HE818423.1和HE818424.1)的亲缘关系较近,遗传漂变是引起烟草花叶病毒不同群体分离物CP基因遗传分化的主要原因。烟草花叶病毒群体分离物CP基因处于负向选择的压力之下,保守性较高,序列的遗传结构较为稳定。研究结果为桂牧一号杂交象草中烟草花叶病毒病的防控及其抗病育种提供一定的理论支持。
【Objective】The paper aimed to identify the main harmful virus species in Pennisetum purpureum cv.Guimu⁃1,to clarify the occur⁃rence and population genetic structure of the virus in P.purpureum cv.Guimu⁃1,and to understand the genetic variation mechanism of the vi⁃rus in P.purpureum cv.Guimu⁃1.【Method】Firstly,Illumina Hiseq 2500 platform was used to perform deep sequencing of small RNAs in P.purpureum cv.Guimu⁃1.Then,E.coli recombinant protein expression and RT⁃PCR technology were used to detect and identify the virus.Fi⁃nally,bioinformatics software was used to analyze codon preference and population genetic structure of the virus.【Result】P.purpureum cv.Guimu⁃1 was detected for tobacco mosaic virus through small RNA sequencing;The recombinant expression of viral proteins in E.coli and the RT⁃PCR results of viral genes confirmed the reliability of small RNA sequencing results;P.purpureum cv.Guimu⁃1 tobacco mosaic virus had six genes,including coat protein(CP),movement protein(MP),charged protein,and so on.The total length of the genes was 11037 bp,with an average length of 1839.5 bp and a GC content of 43.35%in the intergenic region;The codon preference of the CP gene of P.purpu⁃reum cv.Guimu⁃1 tobacco mosaic virus was relatively weak,with a preference for using A/T endings;The internal mutation pressure had a relatively small impact on the codon preference of the CP gene of P.purpureum cv.Guimu⁃1 tobacco mosaic virus,while natural selection had a greater impact on its codon preference;The optimal codons for the CP gene of P.purpureum cv.Guimu⁃1 tobacco mosaic virus were 6(GAA,GCA,GCC,ACU,UUA and AGA),with all codons ending in A and U except GCC;The nucleotide and amino acid sequence consis⁃tency between the CP gene of P.purpureum cv.Guimu⁃1 tobacco mosaic virus and the CP genes of Chongqing tobacco isolates Fengjie⁃1(HE818423.1 and HE818424.1)were high;The CP gene of 10 tobacco mosaic virus isolates from populations including Chongqing,Guan⁃gxi,Guizhou,Hubei,Liaoning,Sichuan,Yunnan,South Korea,Germany and United States had a high level of genetic diversity,with the high⁃est genetic diversity observed in tobacco mosaic virus isolates from the United States,South Korea,and Hubei;The actual observed values of the CP gene mismatch curves of 10 tobacco mosaic virus population isolates all deviated from the expected values,showing a sawtooth shaped peak distribution and being in a dynamic equilibrium state.There had no recent population expansion events among 10 tobacco mosaic virus population isolates;The genetic differentiation of CP genes in 10 tobacco mosaic virus isolates was significant(FST>0.25),and gene ex⁃change was frequent(Nm>1);The dN/dS of the CP gene in 10 tobacco mosaic virus isolates was all much smaller than 1.【Conclusion】The genetic relationship between P.purpureum cv.Guimu⁃1 tobacco mosaic virus and Chongqing tobacco isolates Fengjie⁃1(HE818423.1 and HE818424.1)is relatively close;Genetic drift is the main cause of genetic differentiation of CP genes in tobacco mosaic virus isolates from different populations;The CP gene of tobacco mosaic virus population isolates is under negative selection pressure,with high conservation and a relatively stable genetic structure of the sequence.The results provide certain theoretical support for the prevention and control of tobacco mosaic virus disease in P.purpureum cv.Guimu⁃1 and its disease resistant breeding.
作者
洪森荣
章雪丽
林子贻
林道兴
HONG Sen-rong;ZHANG Xue-li;LIN Zi-yi;LIN Dao-xing(College of Life Sciences,Shangrao Normal University,Shangrao,Jiangxi 334001,China)
出处
《西南农业学报》
北大核心
2025年第6期1150-1161,共12页
Southwest China Journal of Agricultural Sciences
基金
国家自然科学基金项目(31860084、31960079、32060092)
江西省现代农业产业技术体系建设专项(JXARS-13-赣东站)。
